The present invention relates to optical apparatus and, more particularly, to fluorescence observation optical apparatus including a fluorescence microscope and a stereoscopic microscope allowing fluorescence observation.
In recent years, fluorescence observation under a fluorescence microscope and a stereoscopic microscope has been widely used not only in micro observation but also in macro observation at low magnification. In particular, fluorescence proteins such as GFP (Green Fluorescence Protein), CFP (Cyan Fluorescence Protein) and YFP (Yellow Fluorescence Protein) have advantages in comparison to conventional fluorescent dyes. That is, such fluorescence proteins show a comparatively low toxicity to cells, suffer less fading and provide brighter fluorescence. Accordingly, the use of fluorescence proteins in the field of genetic research has also increased.
Thus, objects to be observed range from cells in micro observation to individuals such as fruit flies and mice in macro observation. Therefore, there has been proposed a fluorescence observation apparatus including not only an ordinary fluorescence microscope but also a stereoscopic microscope as an apparatus allowing fluorescence observation.
The stereoscopic microscope is a microscope characterized by having a very long working distance in comparison to ordinary microscopes and allowing three-dimensional observation.
FIG. 43 shows a conventional fluorescence observation apparatus including a stereoscopic microscope. First, the observation optical system of the stereoscopic microscope has an interchangeable objective 41 and two variable magnification optical systems 42R and 42L associated with right and left eyes, respectively. The observation optical system further has imaging lenses 43R and 43L and eyepieces 44R and 44L. An image of a sample 47 is magnified by the objective 41 and the variable magnification optical systems 42R and 42L, and the magnified image of the sample 47 is viewed through the imaging lenses 43R and 43L and the eyepieces 44R and 44L.
The objective 41 and each of the variable magnification optical systems 42R and 42L are arranged in the form of an afocal optical system. Similarly, the variable magnification optical systems 42R and 42L and the imaging lenses 43R and 43L are arranged in the form of afocal optical systems, respectively. Thus, the observation optical system is excellent in system flexibility.
The fluorescence illumination optical system of the stereoscopic microscope has a light source 51, an illumination lens system 52, an excitation filter 53, and a dichroic mirror 54L.
Light from the light source 51, which is a mercury lamp, is led to the excitation filter 53 through the illumination lens system 52. Of the light from the light source 51, only excitation light of wavelength needed to excite the sample 47 is selectively transmitted by the excitation filter 53. Excitation light emanating from the excitation filter 53 is reflected toward the variable magnification optical system 42L by the dichroic mirror 54L and applied to the sample 47 through the variable magnification optical system 42L and the objective 41.
At the sample 47, fluorescent light is produced from portions of the sample 47 stained with a fluorescent dye by illumination with the excitation light. The fluorescent light from the sample 47 is collected by the objective 41 and led to a right observation optical path R for an observer's right eye and also to a left observation optical path L for an observer's left eye. Fluorescent light led to the left observation optical path L passes through the variable magnification optical system 42L and the dichroic mirror 54L and reaches an absorption filter 55L. The absorption filter 55L transmits only fluorescent light of specific wavelength selected according to the spectral characteristics thereof. The fluorescent light of specific wavelength is imaged through the imaging lens 43L and viewed as a fluorescence image through the eyepiece 44L. Fluorescent light led to the right observation optical path R passes through the variable magnification optical system 42R and a dichroic mirror 54R and reaches an absorption filter 55R. Fluorescent light passing through the absorption filter 55R, as in the case of fluorescent light passing through the absorption filter 55L, is imaged through the imaging lens 43R and viewed as a fluorescence image through the eyepiece 44R.
The arrangement of an ordinary fluorescence microscope is shown in FIG. 44. The fluorescence illumination optical system of the ordinary fluorescence microscope has a light source 51, an illumination lens system 52, an excitation filter 53, a dichroic mirror 54, and an absorption filter 55. Light from the light source 51, which is a mercury lamp, is led to the excitation filter 53 through the illumination lens system 52. Of the light from the light source 51, only excitation light of wavelength needed to excite a sample 47 is selectively transmitted by the excitation filter 53. Excitation light emanating from the excitation filter 53 is reflected by the dichroic mirror 54 and applied to the sample 47 through an objective 41. Fluorescent light from the sample 47 is collected by the objective 41 and passes through the dichroic mirror 54 to reach the absorption filter 55. The absorption filter 55 transmits only fluorescent light of specific wavelength selected according to the spectral characteristics thereof. The fluorescent light of specific wavelength is imaged through an imaging lens 43 and viewed as a fluorescence image through an eyepiece 44. The fluorescence illumination optical system shown in FIG. 43 projects an image of the light source 51 in the vicinity of the pupil position of the variable magnification optical system 42L and allows the illumination area and the observation area to coincide with each other independently of a change in magnification made during observation and also independently of interchanging the objective 41 with another objective. Therefore, the fluorescence illumination optical system is excellent in operability.
Similarly, the fluorescence illumination optical system shown in FIG. 44 projects an image of the light source 51 in the vicinity of the pupil position of the objective 41 and is therefore capable of making the illumination area and the observation area coincident with each other independently of interchanging the objective 41 with another objective.
FIG. 45 shows an arrangement in which an observation optical path is not used also as an illumination optical path, unlike the illumination method shown in FIG. 43. The fluorescence illumination optical system shown in FIG. 45 has a light source 51, a collector lens system 58, a light guide fiber 59, an excitation filter 53, and an illumination lens system 57 capable of varying the illumination area. Excitation light from the light source 51 is collected by the collector lens system 58 and led to an entrance end surface 59a of the light guide fiber 59. Light emerging from an exit end surface 59b of the light guide fiber 59 passes through the illumination lens system 57, which is capable of varying the illumination area, and further passes through the excitation filter 53 whereby only light in a specific wavelength region is selected and applied to a sample 47. Fluorescent light from the sample 47 is viewed through an objective 41, variable magnification optical systems 42R and 42L, absorption filters 55R and 55L, imaging lenses 43R and 43L and eyepieces 44R and 44L as in the case of FIG. 43.
FIG. 46 shows the arrangement of an apparatus proposed in WO99/13370, in which a variable magnification optical system in an observation optical system and an illumination optical system are separated from each other.
As shown in part (a) of FIG. 46, the apparatus has an objective 41 and observation optical systems 42L and 42R associated with observer's left and right eyes, respectively, which are provided in an observation optical system unit 42. The apparatus further has imaging lenses 43L and 43R and eyepieces 44L and 44R.
An absorption filter 50 is placed between the observation optical system 42L and the imaging lens 43L. Another absorption filter 50 is placed between the observation optical system 42R and the imaging lens 43R. In a fluorescence illumination optical system 45, as shown in part (b) of FIG. 46, light from a light source 46 is collected and passed through an excitation filter 48. Then, excitation light travels via a deflection member 49 and passes through a fluorescence illumination lens unit 42F provided in the observation optical system unit 42 separately from the observation optical systems 42L and 42R. Then, the excitation light illuminates a sample 47 through the objective 41. Fluorescent light from the sample 47 passes through objective 41 and further through the observation optical systems 42L and 42R and the absorption filters 50 and is viewed through the eyepieces 44L and 44R.
When the magnification for observation is changed by a magnification changing operation of the observation optical systems 42L and 42R in the observation optical system unit 42, lens elements in the fluorescence illumination lens unit 42F move in association with the magnification changing operation of the observation optical systems 42L and 42R to make the observation area and the illumination area coincident with each other. It should be noted that part (c) of FIG. 46 is a top view showing the observation optical systems 42L and 42R and the fluorescence illumination lens unit 42F.
In fluorescence observation, bright and high-contrast fluorescence images are demanded.
Because fluorescent light is very weak in intensity in comparison to light in ordinary reflected-light observation or transmitted-light observation, it is very important to allow a fluorescence image of a sample to be viewed with high brightness and high contrast through not only stereoscopic microscopes allowing fluorescence observation but also various microscopes used for fluorescence observation.
As factors in providing bright fluorescence images, for example, it is demanded that the objective and other associated optical systems should have a high numerical aperture and exhibit a high transmittance over from the ultraviolet region to the visible region, and that illumination efficiency should be increased.
One of the causes of the reduction in contrast of the fluorescence image is autofluorescence, that is, fluorescent light produced from an optical member, e.g. glass, by excitation light. Although a vitreous material producing minimum autofluorescence is selected to form an objective for fluorescence observation, a glass material of high dispersion and high refractive index used as a material of a negative lens, in particular, produces a high degree of autofluorescence and has a low transmittance in the ultraviolet region. Therefore, there is a limitation on the selection of vitreous materials, and it is difficult in terms of optical design to favorably correct aberration of the objective and other optical systems for fluorescence observation.
Accordingly, the stereoscopic microscope shown in FIG. 43 provides an unfavorably dark fluorescence image during fluorescence observation because the numerical aperture is low in comparison to the ordinary fluorescence microscope, although the stereoscopic microscope has the advantageous features that it allows three-dimensional observation and has a long working distance whereby to provide excellent operability. Moreover, because the variable magnification optical system 42L and the objective 41 are placed in the optical path through which excitation light passes, as shown in FIG. 43, autofluorescence occurs from the glass, causing the contrast of the fluorescence image to be reduced unfavorably. Furthermore, because excitation light passes through a long optical path of glass, the degree of autofluorescence occurring in the optical path is very high in comparison to the objective of the ordinary fluorescence microscope. In addition, because the numerical aperture is low, the fluorescence image obtained with the stereoscopic microscope is darker than in the case of the ordinary fluorescence microscope, as stated above. Moreover, the transmittance in the ultraviolet region is low. Therefore, the contrast of the fluorescence image becomes lower than in the case of the ordinary fluorescence microscope.
Thus, it is essential to minimize autofluorescence in order to observe the fluorescence image with high contrast. FIGS. 45 and 46 show arrangements heretofore proposed to solve the above-described problems.
In the fluorescence illumination optical system shown in FIG. 45, because excitation light does not pass through the observation optical path, no autofluorescence occurs in the observation optical system. Accordingly, a high-contrast fluorescence image can be obtained.
However, because the illumination area of the fluorescence illumination optical system does not change in association with the change of the observation area caused by the magnification changing operation of the observation optical system, operability is very bad. Moreover, because a light guide fiber is used in the illumination optical system, the excitation light illumination efficiency is low. Accordingly, the fluorescence image for observation is unfavorably dark.
Furthermore, to perform fluorescence observation with different wavelengths of excitation light, it is necessary to change each of the excitation and absorption filters individually. Accordingly, operability is not good.
The fluorescence illumination optical system shown in FIG. 46 has the observation optical systems 42L and 42R in the observation optical system unit 42, together with the fluorescence illumination lens unit 42F for the exclusive use of the fluorescence illumination optical system. Accordingly, excitation light does not pass directly through the observation optical systems 42L and 42R, and autofluorescence does not occur. However, because excitation light passing through the fluorescence illumination lens unit 42F enters the objective 41, autofluorescence occurs in the objective 41. When the objective 41 is formed by using a vitreous material producing minimal autofluorescence in order to minimize autofluorescence produced from the objective 41, it becomes impossible to maintain the required optical performance, including chromatic aberration correcting performance, in comparison to the optical performance of conventional objectives, as stated above.
In a case where the occurrence of autofluorescence in the objective 41 cannot be suppressed, a region in the objective 41 through which the excitation light passes and a region in the objective 41 through which fluorescent light from the sample 47 passes when it is led to the observation optical systems 42L and 42R overlap each other at a certain region in the observation area. Therefore, autofluorescence light from the objective 41 is superimposed on the fluorescence image in that region. As a result, the contrast of the observation image is partially degraded. Regarding this phenomenon, the region where autofluorescence light is superimposed on the fluorescence image changes according to the magnification of the variable magnification optical systems in the observation optical systems 42L and 42R. Normally, when the zoom ratio of the variable magnification optical systems is low, autofluorescence light is partially superimposed on the observation area. As the zoom ratio increases, autofluorescence light from the objective 41 is superimposed on the whole observation area.
Furthermore, it is conceivable that when excitation light passes through the fluorescence illumination lens unit 42F in the observation optical system unit 42, excitation light reflected from the lens surfaces in the fluorescence illumination lens unit 42F may enter the observation optical systems 42L and 42R in the form of stray light or leakage light. In such a case, if the excitation light causes autofluorescence to occur from the lenses in the observation optical systems 42L and 42R and the absorption filters 50, it is impossible to perform fluorescence observation with high contrast.
Moreover, the observation optical system unit 42 is so structured as to move in association with the movement of the lens units of the observation optical systems 42L and 42R in the observation optical system unit 42 to change the magnification in order to make the illumination area and the observation area coincident with each other. Therefore, it is difficult to spatially shield the fluorescence illumination lens unit 42F to prevent excitation light from entering the observation optical systems 42L and 42R as stray light or leakage light.
Furthermore, the fluorescence observation apparatus having the observation optical system unit 42 cannot be incorporated into a stereoscopic microscope that has heretofore been used; it is used as a special-purpose fluorescence stereoscopic microscope. Therefore, the fluorescence illumination apparatus cannot be used in combination with a stereoscopic microscope that has heretofore been used. Accordingly, the apparatus is inferior in compatibility and system flexibility.
Incidentally, there have been proposed various surgical microscopes in which an observation optical system and an illumination optical system are separated from each other, although they are not stereoscopic microscopes allowing fluorescence observation. FIG. 47 shows an example of the arrangement of a surgical microscope disclosed in Japanese Patent Application Post-Examination Publication No. Hei7-57226.
An affected part 60 to be submitted to a surgical operation is observed through an observation optical system having an objective 61, a variable magnification optical system 62 for observation, a beam splitter 63, an observation prism 64, and an eyepiece 65. The beam splitter 63 is used to branch off a photography optical path from the observation optical path. Thus, the optical path is branched off to a photography optical system (not shown) having an optical axis in a direction perpendicular to the plane of the figure. An illumination optical system for observation that illuminates the affected part 60 has a light source lamp 66, a first relay lens system 67, a variable magnification optical system 69 for illumination, an illumination prism 70, and the objective 61. Light from the light source lamp 66 illuminates the affected part 60 through the illumination optical system for observation. An illumination optical system for photography has a xenon (Xe) flash lamp 71, a second relay lens system 72, a semitransparent reflecting mirror 68, the variable magnification optical system 69, the illumination prism 70, and the objective 61. The semitransparent reflecting mirror 68 is capable of being inserted into and withdrawn from the illumination optical system for observation. To take photographs, the semitransparent reflecting mirror 68 is raised to a position 68′ shown by the dashed lines by, for example, a rotary solenoid (not shown). Consequently, light from the Xe flash lamp 71 illuminates the affected part 60 through the illumination optical system for photography.
The above-described surgical microscope has the features that because the distance to the affected part to be submitted to a surgical operation is long, the working distance is long and the depth of focus is deep in comparison to the stereoscopic microscopes, and further, the zoom ratio of the observation optical system is smaller than that of the stereoscopic microscopes by a factor of about 10. The surgical microscope is unsuitable for fluorescence observation and different from the stereoscopic microscope allowing fluorescence observation in use application and also in performance required of the observation optical system and the illumination optical system.